| BackgroundGastric cancer is one of the four main cancers worldwide. Each year new gastric cancer cases and death cases due to stomach cancer is much more higher among all cancers. It is a serious threat to human health. In 2000, the global newly diagnosed cases of gastric cancer were more than 870,000 and deaths over 640,000. In 2012, the two data were 950,000 and 720,000. More than 60% gastric cancer cases distribution in developing countries, common in China, South Korea and other East Asian countries, also in Japan. In China, gastric cancer is one of the most common malignant tumors with high mortality.Gastric cancer can be caused by a variety of factors. The incidence of gastric cancer is a complex and multi-factors regulating process. Some studies have shown that there is a great relevance between Helicobacter pylori infection and gastric cancer. Environmental and dietary factors also have important roles in the incidence of gastric cancer. In addition, the incidence of gastric cancer has familial aggregation tendency, suggesting that genetic factors also contribute to the occurrence of gastric carcinoma.Carbohydrate antigen 72-4 (CA72-4) is an important glycoprotein tumor marker and has a very high correlation with gastric cancer. CA72-4 is one of the most important tumor markers in clinic. Also, CA72-4 antigen is meant for tumor size, metastasis, staging and predicting of the prognosis of gastric cancer. In clinical practices, CA72-4 are often combined with other markers to improve the sensitivity, specificity and validity of diagnosis of gastric cancer.At present, there are many detection methods for CA72-4. According to the detection targets, we can divide these methods into two types:gene detection and the detection of proteins, including real-time fluorescence quantitative PCR (RTFQ-PCR), electrochemiluminescence immunoassay (ECLIA), Luminescent enzyme immunoassay (LEIA), radio immunoassay (RIA), lateral-flow chromatographic immunoassay and enzyme-linked immunosorbent assay (ELISA). However, these methods have many defects, for example, PCR needs much more time and complex sample processing, also, is easy to pollution. The requirements of operating environment and personnel are high. Moreover, it can not directly detect proteins. Chemiluminescence can directly detect CA72-4 antigen, but it needs long time and large testing equipment, also with high cost. For ordinary lateral flow strips, because fluorescent materials used in strips have short half-life and easy to bleaching, make them unable to realize accurate quantitative testing. Therefore, there is an urgent need for a rapid detection method with accurate quantification and low cost.Immunochromatography (IC) is akind of rapid immunoassay methods which combines immunological techniques and chromatographic technology. Antigens or antibodies in samples to be detected migrate to detection region by capillary chromatography, antigens and antibodiesreact by immune responseto achieve specific detection. The most prominent advantage of immunochromatography is one-step detection of samples without separation of free markers. It is rapid, simple and accurate, widely used in clinical diagnosis, drug testing, food safety detection, detection of plant pathogens and drug residues and so on. IC, which originated in 1980s, was used for the detection of c-reactive protein (CRP) in human plasma by enzymatic coloration. In 1990, colloidal gold was used as marker for the first time to detect human chorionic gonadotropin (hCG) in human urine and serum, pioneering the first application of colloidal gold-labeled lateral flow strips. In recent years, with the development of the studies on preparation and application of novel nanomaterials, a series of new markers, such as colloidal carbon, rare-earth elements, up-converting phosphor (UCP), fluorescent microspheres, magnetic beads, latex particles and quantum dots gradually emerge. These nanomaterials improve and perfect the traditional immunochromatographic assay and laid the foundation for wide application of immunochromatography.Quantum dots (QDs) also known as nanocrystals, is a nanoparticle usually composed by one kind of II-VI, III-V or IV-VI elements. Its size is less than 10 nm. It has obvious quantum effects such as surface effect, confinement effect, tunnel effect and size effect and so on. Quantum dots have a wide excitation spectrum, relatively narrow emission spectrum, very good fluorescence intensity and stability. In addition, emission spectra of quantum dots change when its size and composition are different. These characteristics make quantum dots become a kind of important fluorescent probes for immune analysis.Quantum dots can be formed by one kind of semiconductor materials, such as CdS, CdSe, CdTe, ZnSe etc., also composed of two or more than two kinds of semiconductor materials. Traditionally, quantum dots made by TOP-TOPO organic phase synthesis methods can not be directly used in the fields of biology due to the hydrophobic property. Functional groups or silica should be modified on the surface of QDs to make QDs hydrophilic, and then connect bioactive substances to achieve its biocompatibility. However, these additional modifications will lead to larger size of quantum dots and reduce the fluorescence yield. With the rapid improvement of QDs synthesis technology in recent years, phase synthesis methods gradually emerge. Quantum dots, with various functional groups such as carboxyl, thiol can be prepared. It has laid a foundation of the wide application in the biomedical field.Contents and methodsThis study aims to develop CdSe/ZnS quantum dots-labeled lateral flow strips for rapid and quantitative detection of gastric cancer carbohydrate antigen 72-4 to meet the needs of clinical detection.The main contents and methods include,(1) First of all, make characterizations and identifications of physical and chemical properties of CdSe/ZnS quantum dots, including electron microscopy, identification of excitation spectra and emission spectra to get a kind of water-soluble QDs with 5-7 nm diameter. The QDs has carboxyl (-COOH) and easy to connect with monoclonal antibodies. The quantum dots have strong fluorescence intensity, resistance to bleaching, non-toxic and so on, and emit bright red fluorescence once excited by 365 nm light.(2) The next, screen monoclonal antibodies against CA72-4 antigen and select two monoclonal antibodies CC49 and B72.3 as detection antibody and coating antibody respectively. We also identify their purity and molecular weight.(3) Then, the CdSe/ZnS quantum dots and monoclonal antibody CC49 were coupled to be QDs-CC49 probes. The labeling methods were explored and we found the best conditions. Use BCA method to determine the amount of monoclonal antibody CC49, get the standard curve (y=0.1382x-0.0016, R=0.998), and then calculate the labeling efficiency of quantum dots and CC49. The results show that CC49 and QDs labeled with high efficiency. The results of fluorescence spectrophotometer scanning found that naked quantum dots and QDs-CC49 probes had similar excitation spectra and emission spectra. QDs-CC49 has higher fluorescence intensity. These results indicate that theQDs-CC49 probes have all features of quantum dots and monoclonal antibody and can be used for strips development.(4) Then, place QDs-CC49 probes and monoclonal antibody B72.3 on glass fiber and nitrocellulose membrane respectively to form conjugate pad and reaction membrane and then assemble to rapid quantitative detection strips for CA72-4 antigen based on quantum dots. This study firstly established a QDs based lateral-flow platform with double antibodies sandwich method, including the selection and verification of the qualitative and quantitative detection instruments. Then, make detailed examination of the treatment process and conditions of the strips, especially the coating concentration, time and buffer of monoclonal antibody B72.3 on nitrocellulose membrane, the coating amount of QDs-CC49 on conjugate pad and the recovery of the strips. The study also investigated the homogeneity and stability of the strips.(5) Finally, this study evaluated therapid quantitative detection strips for CA72-4, mainly including three parts:first, analytic sensitivity and specificity were determined using the gradient dilution of CA72-4 antigen standard. Second, detect clinical gastric carcinoma specimens (11 CA72-4 single positive samples, 29 multi-positive samples and numbers of negative specimens) to assess the sensitivity and specificity of the strips. Third, to compare the performance of the strips and Roche ECL kit, we also detect 100 clinical specimens (70 CA72-4 positive samples and 30 negative samples).ResultsIn this study, we labeled CA72-4 monoclonal antibody CC49 onto the surface of CdSe/ZnS quantum dots and then develop rapid quantitative detection strips for CA72-4 antigen based on quantum dots. We studied and established the process parameters and conditions of the strips development, and comprehensively evaluated the sensitivity and specificity of the strips. The results show that the analytic sensitivity of the strips for CA72-4 antigen standard is about 2 IU/mL. The sensitivity and specificity for 11 CA72-4 single positive serum specimens and 6 negative samples are both 100%(11/11 and 6/6). The sensitivity for 29 multi-positive samples and 10 negative serum specimens are both 100%(29/29 and 10/10) too. The results of the detection of 100 clinical serum specimens show the correlation between the strips and Roche ECL kit is 100% (100/100). All the above results suggest that the CdSe/ZnS QDs-labeled lateral flow strips for rapid and quantitative detection of CA72-4 is an acceptable detection test with good performance. The operation of the strips is extremely simple and only takes 5-10 minutes. The strips can play an important role in clinical detection of gastric cancer. |
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